Human TLRs are critical sensors for microbial components leading to the production of proinflammatory cytokines that are controlled by various mechanisms. Monocytes pretreated with LPS exhibit a state of hyporesponsiveness, referred to as cross-tolerance, to both homologous and heterologous ligands, which play a broader role in innate immunity. To date, LPS-induced cross-tolerance has not been examined regarding microRNA expression kinetics. In this study, THP-1 monocytes treated with various inflammatory ligands showed a continuous amplification of microRNA (miR)-146a over 24 h that is inversely correlated to TNF-α production. In contrast, inhibition of miR-146a showed a reciprocal effect. Thus, the characteristic upregulation of miR-146a in LPS-exposed THP-1 monocytes was studied for cross-tolerance. Strikingly, in LPS-tolerized THP-1 monocytes, only miR-146a showed a continuous overexpression, suggesting its crucial role in cross-tolerance. Similarly, peptidoglycan-primed THP-1 cells showed homologous tolerance associated with miR-146a upregulation. Subsequently, interchangeable differential cross-regulation was observed among non-LPS ligands. TLR2 and TLR5 ligands showed both homologous and heterologous tolerance correlated to miR-146a overexpression. More importantly, inflammatory responses to TLR4, TLR2, and TLR5 ligands were reduced due to knockdown of miR-146a targets IL-1R-associated kinase 1 or TNFR-associated factor 6, suggesting the regulatory effect of miR-146a on these TLRs signaling. Transfection of miR-146a into THP-1 cells caused reduction of TNF-α production, mimicking LPS-induced cross-tolerance. Aside from individual ligands, a whole bacterial challenge in LPS-primed THP-1 monocytes was accompanied by less TNF-α production, which is conversely correlated to miR-146a expression. Our studies have thus demonstrated that miR-146a plays a crucial role for in vitro monocytic cell-based endotoxin-induced cross-tolerance.